7alpha - lower alkylthio - 17beta - hydroxy - 17alpha- methyl - 5alpha - androstano(3,2 - c)pyrazole and novel intermediates useful in the preparation thereof



United States Patent US. Cl. 424-241 8 Claims ABSTRACT OF THE DISCLOSURE7a lower alkylthio 17p hydroxy-17a-methyl-5a-androstano[3,2-C] pyrazolesare prepared by reduction of a 7m lower alkylthio17fl-hydroxy-17a-methyl-4 androstan-one with lithium in liquid ammoniathereby producing a 70: lower alkylthio 17fi-hydroXy-l7a-methyl-5aandrostan-3-one. These novel intermediates are reacted with an alkylformate in the presence of a strong base in a non-polar solvent with theproduction of 7a-alky1thio- 17p hydroxy 2 hydroxymethylene 17a methyl50candrostan-B-ones.

These second intermediates are converted to the corresponding 7aalkylthio 17,8 hydroxy-17a-methyl-5a-androstano[3,2-C]pyrazoles byreaction with hydrazine hydrate in ethanol.

The final products have interesting pharmacological properties, withanabolic activity superior to 17,6-hydroxy- 17a methyl 50candrostano[3,2 C]pyrazole and 17- methyltestosterone with lessened sideeffects and lowered acute toxicity.

SUMMARY OF THE INVENTION This is a continuation-in-part of ourapplication Ser. No. 528,809, filed Feb. 21, 1966 and now abandoned.This invention relates to new steroid compounds and intermediates usefulin the preparation thereof. More particularly, the invention relates to7a-lower alkylthio-17flhydroxy-l7a-methyl-5ot-androstano [3,2 C]pyrazoles which possess superior anabolic activity, as well as novelintermediates useful in the preparation thereof, namely, 7u-loweralkylthio-17 3-hydroxy-17a-methyl-5a-androstan- 3-ones and 7a loweralkylthio 17 3 hydroxy 2 hydroxymethylene 17cc methyl 5oz androstan 3ones.

The lower alkyl radicals in these compounds have from 1 to 4 carbonatoms.

Heretofore, it has been known that testosterone and androsterone have ananabolic activity, but they also have an androgenic activity. Therefore,when they are used as an anabolic agent, they show the undesirableandrogenic activity. Efforts have therefore been made to find a newsteroid compound showing enhanced anabolic activity with less androgenicactivity. It has been reported by R. E. Schaub et al. that the originalanabolic activity of a steroid compound was reduced by introducingalkylthio radical at C-7 of the steroid. [R. E. Schaub, M. J. Weiss;

J. Org, Chem, vol. 26, page 3915 (1961)]. See also Schaub et al. US.Patent No. 3,074,932. The anabolic activity of some steroid compounds isincreased by introduction of a pyrazole ring attached to A-ring. [R. 0.Clinton et al.; J. Am. Chem. Soc., vol. 83, page 1478 1961)].

According to the present invention, it has been unexpectedly found that7a-lower alkylthio-17fi-hydroxyl7a-methyl-5a-androstano [3,2-C]pyrazoles show superior anabolic activity when administered orally andsubcutaneously. These compounds possess, therefore, a more favorableratio of anabolic to androgenic activity than that of the parentt-1OWCI' alkylthio steroid compounds.

However, although some analogues of the compounds of this invention havebeen known, for example, in Schaub et al. US Patent No. 3,074,932 citedabove, the compound 17B-hydroxy-7a-methylthio-5a-androstano [3,2-C]pyrazole disclosed in that patent which is most analogous to thecompound of the present invention showed little anabolic activity whenit was administered orally in comparative tests.

Although it has been generally known. that 17-alkylated steroidcompounds impair liver and kidney functions when administered in human,nevertheless the compounds of the present invention do not show suchundesirable side effects.

More particularly, it has been found that the compounds of the presentinvention have a stronger nitrogen retention activity than the otherknown anabolic steroids.

The distinct superiority in the anabolic activity of the compounds ofthe present invention is disclosed in detail hereinafter.

In the comparative tests for anabolic activity, 176-hydroxy-l7a-methyl-5ot-androstano [3,2-C] pyrazole, which has astructure broadly analogous to that of the compounds of the presentinvention and which has been known as a superior oral anabolic agent,and 17a-methyltestosterone, which has been known as an oral anabolicagent, are used as control agents.

Myotrophic and androgenic activity The assay for myotrophic andandrogenic activity was performed according to the modified method ofHershberger et al. [K. Nakamura et al.: Jap. J. Pharmacol., vol. 14,pages 138-149 (1964)].

Male Sprague Dawley rats, 4-5 Weeks old, were used. The animals werecastrated under ether anaesthesia and the oral treatment with testcompounds dissolved into a steroid suspension solution of SV-No. 17874(a standard solvent of the United States Public Health Service) wasstarted on the day of castration and lasted for seven consecutive days.Thetest compounds were administered in doses of 0.2, 0.5, 1.0, 2.0 and4.0 mg./10O g. body weight/day. The animals were killed under etheranaesthesia during the 8th day and the weight of levator ani muscle,ventral prostate and seminal vesicles were determined.

The increase in the weight of the levator ani muscle was used as theindication of myotrophic activity. The androgenic activity wasdetermined from the increase of the weight of the ventral prosta e andseminal vesicles, and Was expressed as the geometrical mean value of thetwo. The relative potencies were estimated graphically. Thesecomparative results are shown in Table I, in which the results werecalculated by the parallel line assay method.

TAB LE I No.0! Myotrophic Androgenlc rats activity activity Nitrogenretention activity The nitrogen retention activity was examined usingmale castrated matured rats 20 weeks old, The animals were given aliquid diet with or with-out addition of the steroid in a metaboliccage. Urinary nitrogen was assayed every day. As summarized in Table II,the representative compound of the present invention, 7a-ethylthio-17Bhydroxy-17u methyl-5a androstano [3,2-C] pyrazole, showed inhibition ofurinary excretion of total It is thus shown that the compounds of thepresent invention have distinctly superior anabolic activity as comparedto l7p-hydroxy-l7u-methyl-5a-androstano [3,- 2-C] pyrazole and17a-methyltestosterone.

In addition, the compounds of the present invention have the leastundesirable side effects, which the known anabolic steroid compoundshave generally shown, such as an androgenic activity and depressions ofpituitary, kidney and liver functions.

The acute toxicity of the compounds of the present invention is verylow; for example LD of the representative compound of the presentinvention, 7a-ethylthio-l7flhydroxy-17a-methyl-5a-androstano [3,2-C]pyrazole, is 1,050 mg./ kg. in male mice and 2,084 mg./ kg. in femalemice.

The subacute toxicity of the compounds of the present invention was alsodetermined using 17x-methyltestosterone as the control. Female and maleDanryu rats, 7 weeks old, were used. The test compounds wereadministered orally with laboratory chow (CLEA, CE-2) in doses of 0.1,1.0 and 10.0 mg./kg./day for 4 consecutive weeks and the influences onbody weight, organ weight, endocrine gland weight, blood picture andleucocyte differential were observed, and macroand microscopicalexaminations of the tissues were performed. In this manner, it wasdetermined that hypertrophy of seminal vesicle: and ventral prostate,and atrophy of pituitary, adrenal,v ovary and uteris was observed onlyin the case of the control drug 17a-methyltestosterone. The compounds ofthe present invention showed only a few instances of atrophy of theuterus in female rats administered in the highest dose of 10.0mg./kg./day.

Thus, the compounds of the present invention are nitrogen, thusmdlcatlng strong nitrogen retentlon. 5 superior anabolic stenod withoutany distinct undesirable TAB LE II Urinary nitrogen excretion BeforeDuring Dose, No. administration administration mg./rat/ o Percent Testcompound day rats mg./rat/day+s.e. retention 7a-ethylthi0-17B 0. 3 5 16.90:1:3. 73 16. 22:1:3. 31 2. 49:1:3. 82 hydroxy-fla- 1. 0 5 21. 31:1:5.12 15. 291:3. 18 23. 72:1:9. 79 methyl-fia-andro- 3. 0 5 20. 425:2. 4314. 69:1:1. 24. 97:1:6. 25 stano [3,2-0] pyrazole. 17fl-hydroxy- 0. 3 519. 99:1:5. 18 16. 06:1:3. 84 4. 86:4:1. 73 17a-methy1-5a- 1. 0 5 20.94:2. 12 17. 42:1:1. 17. 42:1:6: 49 androstano [3,2-C] 3. 0 5 23.94:1:4. 25 18. 45:1:1. 95 16. 12:]:1. 85 pyrazole. 17a-methyl- 0. 3 525. :1:5. 53 30. 24i4. 23 2. 03:]:1. 73 testosterone. 1. 0 5 21. 43:1:1.99 22. 90:1:2. 03 1. 00:1:3. 72 3.0 5 22. 96:1:1. 70 18. 90:1:1. 55 16.89i1. 73

Anabolic incorporation of leucine-UC into tissue proteins side effects.They are useful clinically for the improvement of anabolism, theincrease of body weight, the promotion of growth, the increase ofappetite, the potentiation of physical strength, and the stimulation ofnewgrowth of tissue. In humans, a dosage in the range of 0.5 to 20mg./day may be desirable. More particularly, a dolsage in the range of1.0 to 2.0 rug/day may be desirab e.

The compounds of the present invention may be associated with anytherapeutically acceptable solid or liquid pharmaceutical carrier whichis not incompatible with the active material. Thus, compositionsaccording to the present invention may take the form of tablets,

TABLE 111 D Leucine-U-G into protein-Owing. protein ose, mgJday No. ofPlasma d.p.m./n1g. Muscle, d.p.m./rng. Liver, d.p.m./mg Test compoundp.o. rats protein percent protein percent protein percent Control. 0 5133. 3:1:6. 4 (0) 16. 465:3. 94 (0) 72. 6:|:4. 5 (0)7a-ethylthiod7l9-hydroxy- 0. 5 5 183. 15:19. 4 (+37. 4) 18. 51:1:0. 71(+12. 5) 83. 3:1:4. 6 (+14. 8)

l7a-methyl-5a-androstano [3,2-C] pyrazole. 2. 0 5 157. 75:4. 5 (+18. 4)19. 06:1:0. 81 (+15. 0) 82. 2:1:4. 1 (+13. 2) l7a-methyltestosterone T0. 5 5 146. 2:1:4. 6 (+9. 6) 17. 6410. 68 (+7. 2) 79. 9:1:1. 2 (+10. 0)2. 0 5 78. 9:1:3. 5 (+8. 8)

powders, capsules, syrups or other dosage forms particularly suitablefor oral ingestion.

A preferred pharmaceutical carrier for use in the compositions accordingto the present invention is a lipid, from animal or vegetable sources,either in solid or liquid, form and preferably having a high coefficientof digestibility. Acceptable carriers are oils for example coconut oil,corn oil, cottonseed oil, lard oil, olive oil, peanut oil, sesame oil,soy bean oil, wheat germ oil and egg yolk oil, or fats or butters, forexample butter fat, lard, cocoabutter, margarine fat and the like.Suitable lipid carriers also include monoand diglycerides and synthetictriglycerides.

The intermediate compounds of the present invention, 7a-lOW6Ialkylthio-17p-hydroxy-2-hydroxymethylene-17amethyl-5a-androstan-3-onesare prepared by reduction of 7m loweralkylthio-17fl-hydroxy-l7a-methyl-4-androsten- 3-ones with lithium inliquid ammonia, and by treatment of the resulting novel dihydrocompounds, 7ot-lower alkylthio 17,8 hydroxy17ot-methy1-5u-androstan-3-oneswhich also are intermediate compounds ofthe present inventionwith an alkyl formate, for example ethyl formate,in the presence of a strong base, such as sodium hydride or sodiumalkoxide in nonpolar solvent, for example benzene, at room temperatureor at reflux temperature.

The 7a lower alkylthio 17,8-hydroxy-17ot-methyl-5aandrostano [3,2-C]pyrazoles of the present invention can be prepared by reacting theintermediates obtained above with an equivalent molecule of hydrazinehydrate in alcoholic solution at room temperature or at moderatetemperature.

The following examples illustrate in greater detail the preparation ofthe compounds of the present invention.

EXAMPLE I Preparation of7a-ethylthio-17p-hydroxy-17a-methyl-5aandrostan-B-one A solution of 20g. of 7a-ethylthio-17,8-hydroxy-l7dmethyl-4-androstan-3-one [L M. Krameret al.: Chemische Berichte, Vol. 96, page 2803 (1963)] in 160 ml. ofdioxane and 160 ml. of ether is added dropwise to a solution of 1540 mg.of lithium in 500 ml. of liquid am monia over a period of 30 minutes.After stirring for an additional 40 minutes, the blue color of thesolution is discharged by the addition of 26 g. of dried ammoniumchloride. The excess of ammonia is evaporated at room temperature, theresidue is diluted with water and the solution is extracted with ethylacetate. The ethyl acetate extract is washed with water, dried withanhydrous sodium sulfate, and evaporated to dryness under reducedpressure leaving 33 g. of the residue. The residue is dissolved in 360ml. of methanol and a solution of 90 g. of sodium metabisulfite in 300ml. of water is :added to the methonal solution. After stirring for 30minutes, the resulting solution is diluted with 1500 ml. of water. Theinsoluble material is removed by extracting with ether. To the aqueousphase, 100 ml. of 37% aqueous formaldehyde solution is added and themixture is heated on the waterbath for 30 minutes. The crystallineprecipitate is filtered, washed with water, and dried with anhydroussodium sulfate to give 15.0 g. of the product, melting point 147-150 c.

For analysis, the product is recrystallized from methanol-water, meltingpoint 149-152" C.; [a] 94 (c. 0.1, chloroform); IR 3530 (OH), 1700(C=O).

EXAMPLE II 6 the reaction mixture is allowed to stand overnight and thendiluted with Water to decompose the excess hydride. The resultingorganic layer is separated off and the aqueous phase is washed withbenzene.

The aqueous layer is then acidified. with 5% hydrochloric acid and theprecipitated enol is extracted with ether. The extracts are washed withwater, dried with anhydrous sodium sulfate, and evaporated to dryness togive a crude material. Recrystallization from acetoneether gives 1.55 g.of the crystalline product, melting point 168171 C.; -64.5 (c. 0.9,chloroform);

A 278 mu (6 12100) EXAMPLE III To 0.8 g. of 7u-ethylthio-17 8-hydroxy 2hydroxymethylene-17a-methyl-5ot-androstan-3-one (prepared as describedin Example II) are added 0.255 g. of hydrazine hydrate and 30 ml. ofethanol. The mixed solution is stirred for 4 hours. An excess of Wateris added to the reaction mixture and the precipitate is extracted withether several times. The combined extracts are washed with water, driedwith anhydrous sodium sulfate, and evaporated to dryness to give acrude: material. Recrystallization from acetonehexane gives 0.25 g. ofthe desired product, melting point 151 C.; [a] -60.4 (c. 1.2,chloroform);

max.

224 mp. (e 6500).

EXAMPLE IV Preparation of 17fl-hydroxy-17a-methyl-7a-methylthio-5u-androstan-3-one According to Example I, the product is prepared inthe same way from 17tat-methyl-7a-methylthiotestosterone. The meltingpoint of the product is 201-203 C.; 77.0 (c. 1.6, chloroform).

EXAMPLE V Preparation of 17,8-hydroxy-17tx-methyl-7a-n-propylthio-5a-androstan-3-one According to Example I, the product is prepared inthe same way from 17a-methyl-7u-n-propylthiotestosterone. The meltingpoint of the product is -130 C.; -79.5 (c. 1.9, chloroform).

EXAMPLE VI Preparation of 17B-hydroxy-7a-isopropylthio-17amethyl-Sa-androstan-3 -one EXAMPLE VII Preparation of7a-n-butylthio-17fi-hydroxy-17a-methyl- 5a-androstan-3-one According toExample I, the product is prepared in the same way from7u-n-butylthio-17a-methyltestosterone. The melting point of the productis 114117 C.; [ed 82.6 (c. 1.2, chloroform).

EXAMPLE VIII Preparation of17/8-hydroxy-2-hydroxymethylene-17amethyl-7a-methylthio-5a-androstan-3-one According to Example II, the product is prepared inthe same way from 17p-hydroxy-17u-methyl-7a-methyLthioa5a-androstan-3-one (prepared as described in Example IV). Themelting point of the product is 189192 C.; [11],; 53.2 (c. 1.3,chloroform);

max.

7 EXAMPLE IX Preparation of17/8-hydroxy-2-hydroxymethylene-17amethyl-7a-n-propylthio-5a-androstan-B-One it? 285 9 (e 9,100

EXAMPLE X Preparation of17B-hydroxy-2-hydroxymethylene-7aisopropylthio-17oc-methyl-Sa-androstan-3-oneAccording to Example II, the product is prepared in the same way from17,8-hydroxy-7a-isopropylthio-17amethyl-a-androstan-3-one (prepared asdescribed in Example VI). The melting point of the product is 186188 C.;--69.0 (c. 1.5, chloroform);

A 280 m (e 11,500)

EXAMPLE XI Preparation of7a-n-butylthiol7fl-hydroxy-2-hydroxymethylene-l7a-methyl-5a-androstan-3-oneAccording to Example II, the product is obtained in the same way from7ot-Il-bl1tY1thiO-17B-hYdIOXY-170tmethyl-5u-androstan-3-one (prepared asdescribed in Example VII). The melting point of the product is 187 190C.; 64.9 (c. 1.4, chloroform);

EXAMPLE XII Preparation of l7fl-hydroxy-17a-methyl-7a-methylthio-5a-androstano [3,2-C] pyrazole max.

EXAMPLE XIII Preparation ofI7,8-hydroxy-l7a-methyl-7a-n-propylthio-5a-androstano [3,2-C] pyrazoleAccording to Example III, the product is obtained in the same way from17 8-hydroxy-Z-hydroxymethylene- 17a methyl 7a. n propylthio 5ozandrostan-3-one (prepared as described in Example IX). The melting pointof the product is 135l36 C.; 54.0 (c, 1.9 chloroform);

EXAMPLE XIV Preparation of1718-hydroxy-7a-isopropylthio-17arnethyl-5a-androstano [3,2-C] pyrazoleAccording to Example III, the product is prepared in the same way from17fl-hydroxy-2-hydroxymethylene-7aisopropylthio-17u-methyl-5a-androstan-3-one(prepared as described in Example X). The melting point of the productis 152-155 C.; 54.2 (c, 1.5, chloroform);

XELOH EXAMPLE XV Preparation of 7a-n-butylthio-17 8-hydroxy-17u-methyl-5ot-androstano[3,2-C1PYraZ0le According to Example III, the product isobtained in the same way from7ot-n-butylthio-17fl-hydroxy-2-hydroxymethylene-17a-methyl-5a-androstan-3-one(prepared as described in Example XI). The melting point of the productis l30134 C.; -53.8 (c, 1.0, chloroform);

AEtOH What is claimed is:

1. A compound 7a-lower alkylthio-17fl-hydroxy-17otmethyl-5a-androstano[3,2-C] pyrazole.

2. The compound l7fi-hydroxyl-17ot-methy1-7a-methy1- thio-5a-androstano[3,2-C] pyrazole.

3. The compound 7a ethylthio hydroxy 17amethyl-Sa-androstano [3,2-C]pyrazole.

4. The compound 17;? hydroxy 17a methyl 7a npropylthio-5a-androstano[3,2-C] pyrazole.

5. The compound 17B-hydroxy-7a-isopropylthio-17amethyl-5a-androstano[3,2-C] pyrazole.

6. The compound 7a n -1butylthio 17fl-hydroxy-17otmethyl-5a-androstano[3,2-C] pyrazole.

7. A pharmaceutical composition for oral ingestion comprising as theprincipal therapeutic agent 7a-loweralkylthio-l7B-hydroxy-17a-methyl-5a-androstano [3,2-C] pyrazole and anontoxic lipid carrier.

8. A pharmaceutical composition for oral ingestion comprising as theprincipal therapeutic agent 7u-ethylthio-17B-hydroxy-l7a-methyl-5u-androstano [3,2-C] pyrazole and a nontoxiclipid carrier.

References Cited UNITED STATES PATENTS 3,074,932 1/1963 Schaub et a1260239.5

HENRY A. FRENCH, Primary Examiner US. Cl. X.R. 260239.5, 397.4

